The present invention relates to methods of expansion of renewable stem cells, to expanded populations of renewable stem cells and to their uses. The present invention further relates to therapeutic applications in which these methods and/or the expanded stem cells populations obtained thereby are utilized.
In vitro expansion of hematopoietic stem-progenitor cells (HSPCs) is constrained by default pathways of commitment and differentiation. Exposure of progenitor cells to different combinations of cytokines supports their exit from the G0/G1 phase of the cell cycle and enables extensive proliferation. However, such proliferation is tightly coupled with commitment and differentiation rather than self-renewing division [Avalos et al. (2002) Mol. Cell. 10:523-535]. Uncoupling of these cellular events by genetic manipulations may lead to uncontrolled cell proliferation and eventually to cell transformation [Zheng Blood (2004) 103(9):3535-43].
To overcome these limitations, attempts are constantly made to identify modulators that can create an environment that favors HSPCs self-renewal with only limited differentiation, in vitro [Amsellem Nat. Med. (2003) 9(11): 1423-7].
The sitruin family of enzymes (also termed, SIRs (for Silent Information Regulators); and Sir-2/Sir2) constitute the class III of deacetylases. These enzymes are conserved in many organisms ranging from bacteria to humans. The founding member of this family, yeast Sir2, has been found in multiprotein complexes and is required for gene silencing in yeast. The functions of human sirtuins have not yet been determined. However, recent studies suggest that the human sirtuins may function as intracellular regulatory proteins with mono-ADP-ribosyltransferase activity.
SIRT family members can be recognized in BLAST searches due to the presence of a conserved core of about 203 amino acid (aa) residues. The archaebacterial family members are not much larger than this core, ranging in size from 245 to 253 aa in length. The eubacterial members are more divergent in length, ranging in size from 208 residues (Actinobacillus actinomycetemcomitans) to 299 residues (Streptomyces), with more variation in the N- and C-terminal extensions. Mammalian SIRT2 is a protein not much larger than the largest prokaryotic SIRT protein. It is, however, considerably smaller than the founding member, Sir2, which is 562 residues in length. Human Sir2 comprise 7 isoforms (i.e., SIRT1-7): these forms are similar in size to Hst2 from budding yeast (357 residues). Like Hst2, mammalian hSIRT2 is a cytoplasmic protein [Afsha (1999) Gene 234:161-168; Perrod (2001) EMBO J. 20:197-209; Yang (2000) Genomics 69:355-3695], while hSIRT3 is a mictochondrial protein.
The conserved core of Sir2 proteins (about 203 residues, approximately 24 kDa) folds into an NAD+-binding protein with intrinsic protein deacetylase activity capable of removing the acetyl moiety from the ε-amino group of lysine residues in protein substrates, including the N terminus of histone H4 and the C terminus of p53. This apparent deacetylase activity of SIRT proteins differs from the histone deacetylase (HDAC, classes I and II) activity of other mammalian and yeast HDACs in its insensitivity to trichostatin A (TSA), insensitivity to sodium butyrate, and strict requirement for NAD+ as a cofactor (see FIGS. 1a-b).
The unique catalytic reaction catalyzed by Sir2-like requires the co-enzyme NAD+ (FIG. 1b). In this reaction, nicotineamide is liberated from NAD+ and the acetyl group of the substrate is transferred to cleaved NAD+, generating the novel metabolite O-acetyl-ADP ribose (OAADPr). This final product as well as ADP-ribose, when injected into starfish oocytes or blastomeres, induces a delay or complete blockage of the cell cycle during development [Borra (2002) J. Biol. Chem. 277:12632-12641]. Production of OAAR by SIRT proteins is coupled closely to NAD-dependent deacetylase (NDAC) activity, which raises the possibility that OAAR may act as a second messenger, electing a yet unknown reaction.
The protein deacetylase function presumed to be intrinsic to all SIRT proteins may be their functional commonality. While the chromatin remodeling properties of Sir2 (resulting from histone deacetylation) in the budding yeast may be atypical of SIRT proteins, as might be expected from the fact that SIRT genes exist in prokaryotes that are devoid of histones. Presumably, eukaryotic SIRT proteins all share the NDAC activity, but differ in their cellular function due to general subcellular distribution and specific protein-protein interactions with their acetylated protein substrates, properties that would be unique to each SIRT ortholog and presumably determined by the folding of the N- and C-terminal extensions as Avalos et al. (supra) have recently suggested. It would not be surprising to find functions for mammalian SIRT proteins that supersede chromatin remodeling.
Recently, it has been suggested that the deacetylase activity SIRT family of enzymes is not restricted to histone protein substrates [Buck (2004) 75(6):939-50].
Indeed, a distant homologue of Sir2 called CobB is found in Salmonella typhimurium, which do not have histones, where it can compensate for the loss of the phosphoribosyltransferase, CobT, suggesting a ribosyltransferase activity. Recent findings also support the concept that nonhistone proteins can serve as substrates for Sir2-like proteins in mammalian cells. For example, it has been shown that human Sir proteins deactylate transcription factors, thereby regulating transcriptional activity. hSIRT1 deacetylates the transcription factor p53 and inhibits its activation in response to DNA damage and oxidative stress. Mouse Sir2α deacetylates the TAFI68 subunit of the TATA-box binding protein-containing factor, leading to the repression of RNA polymerase I transcription.
NF-kappaB is responsible for upregulating gene products that control cell survival. SIRT proteins regulates the transcriptional activity of NF-kappaB. SIRT1 physically interacts with the RelA/p65 subunit of NF-kappaB and inhibits transcription by deacetylating RelA/p65 at lysine 310 [Yeung EMBO J. (2004) 23(12):2369-80]. Treatment of cells with resveratrol, a small-molecule agonist of Sirtuin activity, potentiates chromatin-associated SIRT1 protein on the cIAP-2 promoter region, an effect that correlates with a loss of NF-kappaB-regulated gene expression and sensitization of cells to TNFα-induced apoptosis. While SIRT1 was suggested to be capable of protecting cells from p53-induced apoptosis it's activity augments apoptosis in response to TNFα by the ability of the deacetylase to inhibit the transactivation potential of the RelA/p65 protein [Yeung EMBO J. (2004) 123(12):2369-80].
Another example is the Chicken homolog of Sir-2, chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 (CTIP1 and CTIP2) which enhance transcriptional repression mediated by COUP-TF II and have been implicated in hematopoietic cell development and malignancies. The effects of CTIP1 and CTIP2 CTIP2-mediated transcriptional repression, as well as deacetylation of promoter-associated histones H3/H4 in CTIP2-transfected cells, is reversed by nicotineamide, indicating transcriptional control activity of the chicken homolog. Interestingly, the human homolog of yeast Sir2, SIRT1, was found to interact directly with CTIP2 and was recruited to the promoter template in a CTIP2-dependent manner. Moreover, SIRT1 enhanced the deacetylation of template-associated histones H3/H4 in CTIP2-transfected cells, and stimulated CTIP2-dependent transcriptional repression. Finally, endogenous SIRT1 and CTIP2 co-purified from Jurkat cell nuclear extracts in the context of a large (1-2 mDa) complex. These findings implicate SIRT1 as a histone H3/H4 deacetylase in mammalian cells and in transcriptional repression mediated by CTIP2 [Senawong J Biol. Chem. 2003; 278(44):43041-50].
It was suggested that the SIR2 proteins are histone ADP-ribosyltransferases [Tanny (1999) Cell 99, 735-745], histone/protein deacetylases [Landry (2000) Proc. Natl. Acad. Sci. USA 97, 5807-5811], or both [Kirk Proc Natl Acad Sci USA. 2000 Dec. 19; 97 (26): 14178-14182].
This unique and tightly coupled reaction mediated by these enzymes It is well established that many NAD-dependent enzymes (NAD+ glycohydrolases, ribosyltransferases, and ADP-ribosyl cyclases) form a putative oxocarbenium ADP-ribose cation as the direct product of nicotinamide elimination. Given this precedent for oxocarbenium cation formation and the previously observed NAD+-nicotinamide exchange reaction, SIR2 enzymes will likely form a similar intermediate. Interestingly, in the case of the SIR2 enzymes, oxocarbenium cation formation seems to require acetyl-lysine binding (and/or deacetylation). Only in the presence of acetyl-lysine and NAD+ can exogenously added nicotinamide exchange with the enzyme intermediate to reform NAD+.
Two possible chemical mechanisms for catalysis by the SIR2 family have been proposed. In both cases, a putative oxocarbenium ADP-ribose intermediate is formed by the elimination of nicotinamide from NAD+. In the first mechanism, formation of the oxocarbenium is coupled to acetyl-lysine binding or hydrolysis. On acetyl-lysine hydrolysis, enzyme-bound acetate attacks the oxocarbenium cation to produce 1-O-acetyl-ADP-ribose. Alternatively, acetyl-lysine condenses directly with the oxocarbenium cation. This mechanism would imply that acetyl-lysine binding induces the elimination of nicotinamide to form initially the oxocarbenium intermediate. The acyl oxygen of acetyl-lysine condenses with the oxocarbenium cation. A hydroxide ion then attacks this intermediate to form a tetravalent intermediate, Which can collapse to produce 1-O-acetyl-ADP-ribose through the use of enzyme general acid/base catalysis. Although unlikely based on the above arguments, a different isomer of 1-O-acetyl-ADP-ribose may be formed. It was suggest that the reported histone/protein ADP-ribosyltransferase activity is a low-efficiency side reaction that can be explained through the partial uncoupling of the intrinsic deacetylation/acetate ADP-ribosylation reactions. The fact that these enzymes are capable of an NAD+-nicotinamide exchange reaction suggests that the oxocarbenium cation of ADP-ribose is at least partially susceptible to attack by the base nucleophile. However, the proposed oxocarbenium cation of SIR2 enzymes seems to be exquisitely constructed to limit other possible side reactions, such as attack by H2O or by nucleophilic amino acid side chains, which would result in ADP-ribose or protein ADP-ribosylation, respectively. At most, protein ADP-ribosylation that is about 0.1% of the authentic reaction. Also, ADP-ribose was not detected as a primary enzymatic product. It may be possible that some uncoupling of this reaction to yield protein ADP-ribosylation could result from perturbations in native protein structure (partially unfolded protein, mutagenesis, inappropriate reaction conditions) and from extremely high concentrations of an alternate acceptor, such as reactive protein side chains.
It is suggested that 1-O-acetyl-ADP-ribose, a previously unknown molecule, has a unique cellular function or functions that may be linked to SIR2's gene-silencing effects, raising the possibility that 1-O-acetyl-ADP-ribose has an important signaling role in which other enzymes/proteins may use 1-O-acetyl-ADP-ribose to elicit the proper cellular response. Such targets might include ATP-dependent chromatin remodeling enzymes, histone/protein acetyltransferases, or poly(ADP-ribosyl)transferases. It is interesting to note that poly(ADP-ribosyl)transferases use NAD+ to poly(ADP-ribosyl)ate proteins involved in the metabolism of nucleic acids and in the maintenance of chromatin architecture. One intriguing possibility is that 1-O-acetyl-ADP-ribose could bind poly(ADP-ribosyl)transferases and block poly(ADP ribosyl)ation. Moreover, NAD+ levels in cells are inversely affected by the level of protein poly(ADP-ribosyl)ation. Because poly(ADP-ribosyl)transferases and SIR2 enzymes exhibit similar Km values (about 50-70 μM) for NAD+, they may compete for the available NAD+ and oppose each other's function. Recently, the observation has been made that caloric restriction leading to increased life span seems to be linked through an NAD+- and SIR2-dependent pathway, which raises the possibility that 1-O-acetyl-ADP-ribose may play a role in this phenomenon. It is important to note that bacteria do not have histones, and yet they do have SIR2-like proteins with similar activity, as has been shown here for HST2/SIR2/CobB. Thus, histones need not be the only substrates for deacetylation by these enzymes. Identification of the authentic products and the catalytic mechanism of the SIR2-like enzymes has provided the initial basis for understanding the cellular role(s) played by this important family of proteins. [Cakir (2000). Science 289, 2126-2128].
A number of Sir2 catalytic inhibitors are known, displaying various affinities towards the different Sirtuin family members (i.e., cross-species). Examples include Nicotineamide, Sirtinol and derivatives of the same, Splitomicin and derivatives of the same [Bedalov Proc Natl Acad Sci USA. 2001 98(26):15113-8; Hirao J Biol Chem. (2003) December 26; 278(52):52773-82]. Interestingly, SIR inhibitors and HDAC inhibitors exert different biological activities indicating an activity of SIR different then histone deacetylase [Grozinger J. Biol. Chem., Vol. 276, Issue 42, 38837-38843, (2001)].
Biological activity of the SIR family of enzymes has been implicated in metabolism regulation and aging. Sir2 activation was shown to extend the life-span of yeast under calorie restriction. Similar results were obtained in C. elegans were Sir2 was found to regulate life span through activation of the forkhead transcription factor DAF-16, which is required for longevity [Lin (2000) Science 289:2126-2128]. Indeed, inhibition of Sir-2 was suggested for increasing life-span by controlling stress-resistance of cells (see e.g., WO 2004/016726).
Sir2 was also found to interact with acetyl-transferase PCAF and formed a complex with MyoD. Sir2 transfers the acetyl groups carried by DCAF to MyoD in a NAD+-dependent manner. Over-expression of Sir2 inhibits muscle gene expression and differentiation [Anderson (2003) Science 302:2124-2126]. However, the role of SIRs in events associated with stem cell differentiation is yet poorly understood.
Self-renewal of hemopoietic stem and progenitor cells (HPC) both in vivo and in vitro is limited by cell differentiation. Differentiation in the hematopoietic system involves, among other changes, altered expression of surface antigens [Sieff (1982) Blood 60:703]. In normal human, most of the hematopoietic pluripotent stem cells and the lineage committed progenitor cells are CD34+. The majority is CD34+CD38+, with a minority (<10%) being CD34+CD38−. The CD34+CD38− phenotype appears to identify the most immature hematopoietic cells, which are capable of self-renewal and multilineage differentiation. The CD34+CD38− cell fraction contains more long-term culture initiating cells (LTC-IC) pre-CFU and exhibits longer maintenance of their phenotype. and delayed proliferative response to cytokines as compared to CD34+CD38+ cells. CD34+CD38− can give rise to lymphoid and myeloid cells in vitro and have an enhanced capacity to repopulate SCID mice [Bhatia (1997) Proc Natl Acad Sci USA 94:5320]. Moreover, in patients who received autologous blood cell transplantation, the number of CD34+CD38− cells infused correlated positively with the speed of hematopoietic recovery. In line with these functional features, CD34+CD38− cells have been shown to have detectable levels of telomerase.
Based on the above descriptions, it is clear that there is thus a widely recognized need for, and it would be highly advantageous to have, a method of propagating large numbers of stem cells in an ex-vivo setting. Methods enabling ex-vivo expansion of stem cell compartments yielding large numbers of these cell populations will therefore pioneer feasible stem cell therapies for human treatment, with a clear and direct impact on the treatment of an infinite number of pathologies and diseases. Some pathological and medically induced conditions are characterized by a low number of in-vivo self or transplanted renewable stem cells, in which conditions, it will be advantageous to have an agent which can induce stem cell expansion in-vivo.